Reading assignment: Robbins Pathologic Basis of Disease, 6^th Edition, Chapter 6, pp. 165-176; Chapter 8, p 285

Goals and Objectives

1. Outline how chromosomes are obtained from tissue
2. Describe the normal human chromosome complement
3. Interpret standard chromosome nomenclature
4. Diagram how aneuploidy occurs by nondisjunction
5. List the clinical features of trisomy 21, 18, and 13
6. Explain how Down syndrome can recur in families
7. List the clinical features of sex chromosome abnormalities
8. Categorize several types of structural chromosome abnormalities as balanced or unbalanced
9. Know the common karyotypes in spontaneous abortions
10. Explain how FISH analysis works and what it can be used for, including an example of interphase FISH and of metaphase FISH
11. Describe the uses of cytogenetic and FISH analysis using chronic myelogenous leukemia as an example
12. Be able to order FISH or cytogenetics analysis as appropriate for the specimen and patient situation

Outline

WHAT IS CYTOGENETICS AND WHY DO IT?

nCytogenetics is the study of chromosomes

ÐChromosomes are nuclear structures containing DNA and proteins

nChromosome abnormalities are important mechanism of disease, for example:

ÐEtiology of 50% of spontaneous abortion

ÐChromosome abnormality in 1:160 liveborns

ÐChromosome abnormalities occur in most cancers

ETIOLOGY OF BIRTH DEFECTS/ Pie Chart

TWO MAIN TESTS

nRoutine cytogenetic analysis

ÐIn use since 1960s

ÐLooks at all chromosomes

ÐServes as general screen

nFluorescence in situ hybridization (FISH)

ÐIn clinical use for about 10 years

ÐOnly looks at chosen region(s) of the genome

ÐCome back to this later

ROUTINE APPLICATIONS OF CYTOGENETIC ANALYSIS

nConstitutional analysis--examines chromosome content of all body's cells

ÐPrenatal analysis

ÐPostnatal analysis of babies, kids, and adults for explanation of abnormal phenotype

ÐReproduction issues in adults

nAcquired abnormalities--anomalies found only in tumor tissue

ÐDiagnosis, prognosis, and disease monitoring

STEPS IN CHROMOSOME PREPARATION

Tissue-Culture-Harvest-Slide Making-Banding & Staining-Exam on Scope

WHAT CHROMOSOMES LOOK LIKE

nChromosomes look like this down a microscope after preparation at metaphase

nEach rod is a duplicated structure with constriction called centromere

ÐSite of spindle attachment to assort chromosomes to daughter cells

NORMAL HUMAN COMPLEMENT

nMust know normal to tell what is abnormal

n23 pairs of chromosomes

Ð22 autosomes and 1 pair of sex chromosomes

ÐNormally get 1 of each pair from each parent

nNormal person is diploid--46,XX female or 46,XY male

ÐSperm and egg cells are haploid (23,X or 23,Y)

CHROMOSOME ANALYSIS

nEach chromosome is unique

nChromosomes are organized into a karyotype on basis of:

ÐSize from largest to smallest

ÐPosition of centromere (constriction where microtubules attach)

ÐPattern of light and dark bands induced by trypsin treatment

n400 to 1200 bands per haploid set depending on stage of mitosis

NORMAL MALE KARYOTYPE-46,XY

STANDARD NOMENCLATURE

nInternational standing committee agrees on nomenclature to describe abnormalities and identify each band (based on drawings or ideograms)

nStandard nomenclature allows every cytogeneticist to understand exactly what abnormalities are present regardless of language

X CHROMOSOME DISEASE MAP--FIGURE 6-21

CHROMOSOME 18 IDEOGRAMS

nBands coalesce as they progress through metaphase

n400-550 bands are most common levels used for routine analysis

NOMENCLATURE EXAMPLES

nNumber of chromosomes first, then sex chromosome constitution, followed by any abnormalities in numerical order

ÐEach part separated by commas

n46,XX is a normal female karyotype

n47,XY,+21 is a male with extra chromosome 21

n46,XX,t(9;22)(q34;q11.2) is a female karyotype with a translocation between chromosomes 9 at band q34 and chromosome 22 at band q11.2

PLOIDY

nEuploidy is 23n--23, 46, 69, 92

nAneuploidy is any other number

ÐExtra and missing chrs occur mostly by nondisjunction

nFailure of chromosomes to divide properly at meiosis

nAneuploidy most commonly occurs in maternal meiosis

Ð80% (70% at MI, 10% at MII)

ÐFrequency increases with increasing maternal age

CONSTITUTIONAL NUMERICAL ABNORMALITIES

nDisomy is 2 copies of a chromosome (normal #)

nTrisomy is 3 copies of a chromosome

ÐExample: 47,XX,+21 (Down syndrome)

nMonosomy is 1 copy of a chromosome

ÐExample: 45,X (Turner syndrome)

n Polyploidy

ÐTriploidy, 3 sets of chromosomes

n2 from 1 parent and 1 from the other

nExample: 69,XXY or 69,XXX (spontaneous abortions)

ÐTetraploidy, 4 sets of chromosomes

nExample: 92,XXXX or 92,XXYY

nSpontaneous abortions or some specialized cells in body

STRUCTURAL ABNOMALITIES

nInstead of involving copy numbers of whole chromosomes, structural anomalies involve breakage within a chromosome or chromosomes & rejoining ends in new way

nFall into 2 categories

ÐBalanced

nAll genetic material present, just in different spot

nUsually normal phenotype

ÐUnbalanced

nLoss or gain of genetic material

TYPES OF STRUCTURAL ABNORMALITIES-FIGURE 6-26

ANEUPLOIDY VIABILITY

nOnly viable aneuploidies are +21, 45,X, 47,XXY, and other sex chromosome variations

nTrisomy 13 and trisomy 18 also occur in newborns, but these babies usually die within days after birth, rarely live 1 year

nMosaic trisomy 8 and 9 are also viable

nNo other non-mosaic aneuploidies are found in liveborns

47,XX,+21 KARYOTYPE

DOWN SYNDROME--47,XX,+21

nMost common abnormality in man (1:660 newborns)

nMost common clinical features include:

ÐHypotonia, floppy

ÐFlat face

ÐUpslanting eye slits

ÐExcess neck skin

ÐHeart disease

ÐShort stature

ÐIQ 30-60

Ð10 X increased risk of acute leukemia

KAROTYPIC VARIATION IN DS

nAbout 95% are trisomy 21

ÐIncreased maternal age is greatest risk factor

nAbout 1% are mosaics

ÐMixture of cells with normal karyotype and cells with extra chr 21

(47,X-,+21/46,X-)

ÐMitotic error in embryo

nAbout 4% are translocation DS

ÐHigher risk of recurrence if carried by parent

ROBERTSONIAN TRANSLOCATIONS

nSome members of normal population carry Robertsonian translocations

ÐSpecial type of translocation

ÐRobertsonian translocations involve 2 of chromosomes 13, 14, 15, 21, and 22

nEven though normal person who carries an RB has only 45 chromosomes, considered balanced abnormality in such a person

nAcrocentric chromosomes (13, 14, 15, 21, and 22) have only repeated DNA on short arms

der(14;21)

nTranslocation is not completely reciprocal since short arms are lost and 2 long arms are joined together

nIndividual who carries Rb t has 45 chromosomes, but is normal

TRANSLOCATION DOWN SYNDROME

nNormal individuals who carry Rb ts are at greater risk of having child with:

ÐDS if Rb t involves 14 and 21

Ð+13 if it involves 13 and 14

ÐReason all DS and trisomy 13 cases have to be karyotyped, even if clinically evident, because of risk of recurrence for parents

TRISOMY 18

nClinical features include:

ÐClenched fists

ÐRocker bottom feet

ÐCardiac defects

ÐSevere MR

ÐLimited survival

ÐCan look quite normal

nIncidence 1 in 7500

TRISOMY 13

nClinical features include:

ÐMidline facial defects such as cleft palate and lip, microphthamia or cyclopia

ÐCardiac defects

ÐRocker bottom feet

ÐPolydactyly

ÐSevere MR

ÐLimited survival

nKaryotypes include +13 and Robertsonian der(13;14)

nFrequency 1 in 15-20,000

SEX CHROMOSOME ABNORMALITIES

nAbnormalities of X and Y chromosome have milder effects than analogous abnormalities of autosomes

ÐX chromosome inactivation (lecture 2)

ÐSmall # of genes on Y (lecture 2)

nEffects largely related to sexual development and reproduction

nDiagnosis is often at puberty or in adulthood

45,X KARYOTYPE

TURNER SYNDROME--45,X

nClinical features include:

ÐShort stature

ÐGonadal dysgenesis

ÐWebbed neck, lymphedema

ÐNormal IQ, some LD

nFrequency about 1:5000 female births

ÐDetected at birth or puberty

nVery lethal in utero

nCytogenetically variable

ÐOnly 55% are 45,X

47,XXY KARYOTYPE

KLINEFELTER SYNDROME--47,XXY

nClinical features include:

ÐLong limbs

ÐSmall genitalia

ÐSterility due to azoospermia

ÐSome have enlarged breasts (gynecomastia)

ÐNormal IQ, some LD

nOften detected as adults in infertility clinics

nFrequency is 1:1000 males

nOther karyotypes include:

Ð47,XXY/46,XY

Ð47XXY/48,XXXY

nMore severe

XYY

nFrequency of 1:1000

nNormal phenotype

ÐMay be tall

nNormal IQ

nLow frequency of behavior difficulties

ABNORMALITIES IN SPONTANEOUS ABORTIONS

nMonosomies other than 45,X are rarely found

-Loss of material is more severe wrt phenotype

nAll trisomies are found in SABs or pre-implantation studies

nMost common findings in SABs

Ð45,X

Ð69,XXX or XXY (triploidy)

ÐTrisomy 16

TRIPLOIDY--69,XXY

nCommon in spontaneous abortion, rarely liveborn

nCommon origin is 2 sperm fertilizing 1 egg

DELETIONS

nAll deletions are unbalanced

nClassic deletions of >4 MB

ÐVisible by routine cytogenetics

ÐCri du chat and Wolf Hirshhorn syndromes

nMicrodeletions of ~1-5 Mb

ÐAre difficult or impossible to see cytogenetically

ÐFISH is required to document

ÐPrader Willi, Angelman, and DiGeorge syndromes (more later)

DELETIONS AND DUPLICATION IDEOGRAMS

WOLF HIRSCHHORN SYNDROME

nDeletion of distal end of chromosome 4 short arm (4p16)

nClinical features include:

ÐSevere growth retardation

ÐSevere MR

ÐMicrocephaly

Ð"Greek warrior helmet"

nUsually detectable cytogenetically but may require FISH

RING CHROMOSOME 20 PARTIAL KARYOTYPE

nBreaks at each end and rejoining of portion connected to centromere; loose ends lost

nVery little material missing

nPhenotype in this girl was uncontrolled seizures

nAbout 20 similar cases in literature

LIMITS OF CYTOGENETICS

nCytogenetics is a good general screen for abnormalities if you don't know what you're looking for

nCan't analyze non-dividing cells

nOne chromosome band contains at least 3-5 million base pairs

ÐEven alterations of this size can be difficult to see or diagnose with confidence

HOW BIG IS A CHROMOSOME BAND?

3 X 10⁶ base pairs, ~30 genes

FISH ANALYSIS

nTechnique of fluorescence in situ hybridization circumvents some of limitation of routine chromosome analysis

nSpans part of the resolution gap between cytogenetic analysis and DNA methods, also called molecular cytogenetics

nFISH works on metaphase or interphase cells

FISH PROCEDURE

nMuch like Southern blot

ÐTarget is interphase or metaphase cells on a slide rather than blot

ÐProbe is any DNA sequence available commercially (or home brew), labeled with fluorescent tag

ÐDenature probe and target, then hybridize

ÐDetect on fluorescent microscope

PROBE TYPES

nCentromere probes (alpha satellite, repeated sequence)

nChromosome paint

ÐProbes from an entire chromosome or probe from each chromosome to label all chromosomes, (latter is mainly research)

nUnique sequence

ÐKnown gene

ÐAnonymous sequence

X/Y FISH PROBES ARE REPEATED SEQUENCES

XY FISH

nUsing DXZ1 (X centromere, red) and DYZ1 (Yq12 heterochromatin, green)

ÐInterphase cells (left) identified as male (1R1G) or female (2R)

ÐMetaphase cell (right) shows location of X and Y signals

FETAL TRANSLOCATION-t(3;9)

INHERITED TRANSLOCATION

nFound a translocation of chromosome 9 material to chromosome 3 in a fetus

nStudied parents' blood and found that mother had same translocation

nImplies translocation does not involve loss of material or a break in a gene

nPredict child will be normal

CHROMOSOME PAINT

nUse of a probe from sequences of chromosome 9 "paints" that chromosome

nProves origin of material

SPECTRAL KARYOTYPING

CHROMOSOME 22 MICRODELETION

nVarious phenotypes, different manifestation of same genetic defect

n2 Syndromes

ÐDiGeorge syndrome

nCalcium and thymus defects

ÐVelocardiofacial syndrome

nProminent nose with square nasal root

nCleft palate

n Isolated heart defects

Ð5% of all congenital cases

CHROMOSOME 22 MICRODELETIONS

nVariable expressivity

ÐSome individuals with deletion may have little manifestation

ÐChildren's risk to inherit 50%, can be more severe

nVery important to diagnose

nDifficult to impossible cytogenetically

nFISH is gold standard for diagnosis

DIGEORGE FISH PROBES

D22S275 DELETION BY FISH IN DIGEORGE SYNDROME

nSingle copy sequence from chromosome 22 and control probe marking distal end of 22

nOne chr 22 lacks D22S275 signal

SNRPN LOCUS 15Q11.2

SNRPN HYBRIDIZES TO EXTRA MARKER CHROMOSOME

nSNRPN is a single copy gene on chr 15 labeled in red

ÐChromosomes stained with DAPI

nFISH identifies extra chromosome as 15 derived

nNote 3 copies in interphase nucleus

nPhenotype-autism

CANCER IS A GENETIC DISEASE

nAbnormal accumulation of cells occurs in cancer because of imbalance in genes involved in cell proliferation and death

nChromosome alterations reflect those changes

nSpecific abnormalities occur in specific tumors

ÐTrue for all tumor types, but hematologic disorders most studied for technical reasons

ACQUIRED ABNORMALITIES

nSince specific anomalies occur in specific neoplastic tissues, identification of abnormality can aid in:

ÐDiagnosis

ÐPrognosis

ÐDisease monitoring (remission and relapse)

ÐGene identification and mapping

CANCER CYTOGENETICS

nSame basic types of anomalies occur as in constitutional abnormalities

ÐNumerical and structural, but often multiple and complex

nFirst changes to occur are thought to be primary and later ones secondary or noise

nChanges are clonal--occur in a single cell

nDisappear in remission and recur in relapse

CHRONIC MYELOGENOUS LEUKEMIA

nUsually disease of middle age, incidence of 1-2 per 100,000

nClonal expansion of hematopoietic progenitor cells that increases myeloid and erythroid cells and platelets

nHas chronic, accelerating, and acute phases

BCR-ABL REARRANGEMENT

nABL (Abelson) is a proto-oncogene encoding tyrosine kinase (signal transducer) on chromosome 9; BCR (breakpoint cluster region) is phosphoprotein on 22

nBringing these 2 sequences together is primary event in generation of CML

BCR-ABL REARANGEMENT

nRearrangement accomplished by breaks in ABL and in BCR and rejoining to opposite chromosome, translocation

nTranslocation [t(9;22)] of ABL to BCR generates new fusion protein with increased tyrosine kinase activity

t(9;22) OF CML-IDEOGRAM AND KARYOTYPE

DIAGNOSIS OF CML

nDemonstration of BCR/ABL rearrangement required for diagnosis of CML

nCytogenetic demonstration of t(9;22)

nFISH using probes for BCR and ABL demonstrates fusion

BCR/ABL INTERPHASE FISH

THERAPY BASED ON MOLECULAR ABNORMALITY

n1st cancer chromosome abnormality discovered--Philadelphia chromosome (1960); appreciated as translocation [t(9;22)] in 1973

nFirst "designer drug" therapy for CML based on that abnormality--Gleevec

ÐSignal transduction inhibitor

ÐSome patients on Gleevec show reduction to absence of BCR/ABL fusion on FISH and/or chr analysis

ÐMay turn CML into a truly chronic disease

CYTOGENETICS VS FISH